Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Biological method capable of increasing resveratrol accumulation amount

A technology for resveratrol and the highest yield, applied in the field of bioengineering, can solve the problems of lack of cofactors and low enzyme activity, and achieve the effect of increasing production

Inactive Publication Date: 2019-04-12
CHINA PHARM UNIV
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the production methods of resveratrol mainly include solvent extraction, enzymatic extraction, supercritical CO 2 Extraction, microwave extraction, bio-fermentation extraction, etc., as a method that has emerged in recent years, bio-fermentation has the advantages of low energy consumption, low cost, fast production rate, mild reaction conditions, etc. However, due to the low enzymatic activity of resveratrol synthase, the lack of cofactors in the biosynthesis process has become a limiting factor for the high yield of resveratrol

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Biological method capable of increasing resveratrol accumulation amount
  • Biological method capable of increasing resveratrol accumulation amount
  • Biological method capable of increasing resveratrol accumulation amount

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Construction of two expression plasmids

[0052] 1.1 Primer design

[0053] According to the Rgtal, Pc4cl and Ahsts sequences reported by NCBI, the following 6 pairs of primers were designed:

[0054] Rgtal-F: 5′C CCATGG GCATGGCGCCTCGCCCGACTTCG 3′

[0055] Rgtal-R: 5′CGC GGATCC TGTGCCAGCATCTTCAGCAGAAC 3′

[0056] 4CL-STS-1F: 5′ CATATG GGCGATTGCGTGGCC 3′

[0057] 4CL-STS-1R: 5′GCCAGCGGCGATCTGCCGAAA GGAAGCGGA ATGGTTAGCGTGAGTGGTATC 3′

[0058] 4CL-STS-2F: 5′GATACCACTCACGCTAACCA TTCCGCTTCC TTTCGGCAGATCGCCGCTGGC 3′

[0059] 4CL-STS-2R: 5′ CTCGAG TTAAATGGCCATGCTAC 3′

[0060] 4CL-STS-1NF: 5′GTATAAGAAGGAGATATA CATATG GGCGATTGCGTGGCC 3′

[0061] 4CL-STS-2NR: 5′CGGTTTCTTTACCAGA CTCGAG TTAAATGGCCATGCTACGC 3′

[0062] AccBC-F: 5′ATAAGGAGATATA CCATGG GCGTGTCAGTCGAGACTAGGAAG 3′

[0063] AccBC-R: 5′GCCGAGCTCGAATTC GGATCC TGCTTGATCTCGAGGAGAAC 3′

[0064] PTDH-F: 5′ GCAGATCTCCAATTG GATATC GATGCTGCCGAAACTGGTCATC 3′

[0065] PTDH-R: 5′CTTTACCAGAC...

Embodiment 2

[0081] Example 2 Construction of fumB gene knockout strain

[0082] 2.1 Primer design

[0083] A total of 4 primers are needed to knock out fumB gene, primers S1 and S2 for homologous recombination and primers S3 and S4 for identifying defective strains.

[0084] S1: 5'ATGTCAAACAAACCCTTTATCTACCAGGCACCTTTCCCGAAGCGATTGTGTAGGCTGGAG

[0085] S2: 5'TTACTTAGTGCAGTTCGCGCACTGTTTGTTGACGATTTGCACGGCTGACATGGGAATTAG

[0086] S3: 5'CCTGCGCTATTCACAGAATG

[0087] S4: 5'ATTGCAGCAGATCCACGTAG

[0088] 2.2 Construction of gene-deficient strain ΔfumB

[0089] The homologous recombination helper plasmid pKD46 was transformed into the host strain E.coli BL21(DE3), induced by L-arabinose to prepare electroporation competent cells.

[0090]The plasmid pKD4 containing the kanamycin resistance gene kana was used as a template, and S1 and S2 were used as primers for PCR amplification. Add 2 μL of the resistant fragment pKD4-kana to electroporation competent cells on ice, mix well and place in a 1 m...

Embodiment 3

[0093] Example 3 Transformation of expression plasmids to synthesize resveratrol strains

[0094] The ΔfumB gene-deficient strain was made into a ΔfumB gene-deficient E. coli competent according to the standard procedure for E. coli competent production, and the successfully constructed pCDFduet-Rgtal-Pc4cl-Ahsts and pACYCduet-AccBC-PDTH recombinant plasmids were transformed into the gene-deficient strain ΔfumB, And spread a solid LB plate containing spectinomycin and chloramphenicol in one thousandth, and cultivate at 37°C until the transformants grow out. Among them, the gene-deficient strain ΔfumB is a strain without spectinomycin and chloramphenicol resistance, and cannot grow on solid LB plates containing spectinomycin and chloramphenicol. Therefore, pCDFduet-Rgtal-Pc4cl containing pCDFduet-Rgtal-Pc4cl was obtained by screening with two antibiotics. - Gene-deficient strains of the Ahsts and pACYCduet-AccBC-PTDH recombinant plasmids. Designated as pCDF-ΔfumB-Res

[0095]...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a gene-defective-type Escherichia coli strain containing a resveratrol synthase system and a malonyl coenzyme-A synthase system and a construction method thereof, as well as amethod for increasing resveratrol accumulation amount. A recombinant plasmid composed of the resveratrol synthase system and the malonyl coenzyme-A synthase system provided by the invention includes Rgtal, Pc4cl, Ahsts, accBC and PTDH sequences as well as two suitable vector fragments. The biological method capable of increasing resveratrol accumulation amount provided by the invention comprises the following steps: transforming the recombinant plasmids composed of the resveratrol synthase system and the malonyl coenzyme-A synthase system, namely pCDF-Rgtal-Pc4cl-Ahsts and pACYC-accBC-PTDH, into Escherichia coli strains with fumarinase gene deficiency (delta-fumB) so as to obtain engineering strains; and then, carrying out culturing with an optimized medium. Thus, yield of resveratrol withthe optimized medium can be up 22.62 mg / L, which is increased by 5.73 times compared with yield of original strains.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method for high-yielding resveratrol by a resveratrol synthetase system and a fumaric acid gene B-deficient strain. Background technique [0002] Resveratrol (Res) is a class of polyphenolic compounds containing a stilbene structure, also known as stilbene triphenols, and its chemical formula is C 14 h 12 o 3 , colorless needle-like crystals, easily soluble in ether, methanol, ethanol and other organic solvents, first extracted from the root of Veratrum tomentosa in 1940. Resveratrol is found in many plants in nature, such as grapes, knotweed, peanuts, mulberry plants, etc. It is an anti-toxic substance produced by plants to protect themselves from being attacked by pathogens. Resveratrol has various physiological activities such as prevention and treatment of atherosclerosis, reduction of platelet aggregation, and anti-virus. It has been listed as a health...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/21C12N15/70C12P7/22C12R1/19
CPCC12N15/70C12P7/22
Inventor 许激扬卞筱泓王郑隆
Owner CHINA PHARM UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com